The burgeoning size of the liver transplant waiting list has forced clinicians to increasingly accept organs from marginal donors, which unfortunately are more susceptible to preservation injury and graft dysfunction. The dysfunction appears to be related in part to the invariable cell swelling associated with cold ischemia and reperfusion (CIR) injury incumbent with current organ retrieval and implantation. Elevated cytosolic and mitochondrial calcium is a significant factor in cold ischemic injury to hepatocytes, as it is associated with cellular and mitochondrial swelling; the latter of which results in cytochrome c release and initiation of cellular apoptotic cascades. We have recently described the presence of 2 mitochondrial P2Y purinergic receptors, mP2Y1 and mP2Y2 that alter mitochondrial Ca2+ uptake in response to changes in cytosolic [ATP]. During cold ischemia, cytosolic [ATP] is depressed. Thus, the presence of mitochondrial purinergic receptors suggests a novel regulatory mechanism of mitochondrial Ca2+ in cold-ischemic injury. The hypothesis of this application is that decreased mitochondrial P2Y receptor activation in cold ischemia causes increased mitochondrial Ca2+ uptake and cytochrome c release. To study this hypothesis, we propose the following 2 specific aims. In specific aim 1, the pharmacological characteristics of these receptors in isolated rat liver mitochondria will be established. By using specific agents known to stimulate and antagonize P2Y1 and P2Y2, mitochondrial 45Ca2+ uptake will be measured to determine mitochondrial receptor agonist profiles. Establishing a profile for these receptors will define their probable triggers during CIR. In specific aim 2, the mechanism by which mitochondrial P2Y receptors regulate mitochondrial Ca2+ uptake and cytochrome c release will be determined. By simultaneous antagonism of mitochondrial Ca2+ management pathways and P2Y stimulation, the mechanism by which these receptors alter mitochondrial Ca2+ uptake will be defined. The significance of the proposed research is that it will elucidate a novel pathway involved in the CIR injury, and may also lead to the development of new treatment strategies to reduce CIR injury and graft dysfunction following liver transplantation.